PROJECT SUMMARY Rearrangements of the MLL gene at chromosome 11q23 portend a poor prognosis in patients with acute leukemia. MLL rearrangements are found in 5-10% of acute leukemias and are particularly common both in babies with leukemia and in chemotherapy-associated secondary leukemia. New approaches to the treatment of MLL leukemia are warranted as increasingly aggressive therapy has yielded only modest improvements in survival. Reciprocal translocations at the MLL locus are heterogeneous, but the two most common translocation partners are the AF4 and AF9 genes. We previously demonstrated that AF4 and AF9 proteins form a multiprotein complex. Moreover, the protein complex can be disrupted in vitro and in vivo by small synthetic peptides that mimic the AF9 binding site of AF4. When exposed to one such peptide, designated PFWT, leukemia cell lines expressing MLL-AF4 or MLL-AF9 fusion genes undergo programmed cell death by necrosis. Importantly, no effect on bone marrow colony formation is observed at peptide concentrations that are toxic to leukemia cells. One possible explanation for these observations is that macromolecular complexes comprised of AF4 and AF9 chimeric proteins are crucial for the survival of t(4;11) and t(9;11) leukemia cells. By binding AF9 and disrupting MLL-AF4-AF9 (or MLL-AF9-AF4) protein complexes, PFWT inhibits MLL leukemias. This research project focuses on the role of the mutual interaction domains of AF4 and AF9 and will examine the requirement for binding of AF9 to AF4 or to Dot1 histone methyltransferase in leukemogenesis. By analyzing the contributions of specific proteins to the activity of chimeric oncoproteins, these studies are designed to reveal promising new approaches to treat MLL leukemia. We have devised the PFWT peptide as a prototype of molecules that block protein interactions critical to leukemia cell survival. However, we also hope to extend the impact of experimental tools to feasible treatment strategies. Thus, a highly substituted and more potent derivative of PFWT peptide, SPK- 107, will be tested in a mouse model of human MLL leukemia. These pre-clinical studies are intended to lay the groundwork for pharmaceutical drug development.